Dithiocarbamate tumor-inhibitory process



Unite States atet 3,051,625 DITHIOCARBAMATE TUMOR-ITORY PROCESS Koppaka V. Rao, Pine Brook, N.J., assignor to Chas. Pfizer & (30., Inc., New York, N.Y., a corporation of Delaware No Drawing. Filed July 1, 1959, Ser. No. 824,170

Claims. (Cl. 167-78) This invention relates to a method for inhibiting the growth of tumors and to a composition useful in such treatment. More specifically, it relates to a method for inhibiting malignant tumor growth in animals, including man.

Although the field of cancer therapy has been the subject of intensive study, particularly in recent years, so far extremely few effective substances have been found. Any extension of the range of available agents therefore represents a significant contribution to the knowledge of this important subject.

It has now been discovered that a class of compounds previously believed to be inactive actually possesses substantial tumor inhibitory activity. These valuable agents are the physiologically tolerable salts and esters of N,N- disubstituted dithiocarbamic acid. More specifically they are dithiocarbamates of the general formula where M, may be an alkali metal ion, a radical derived from a pharmaceutically acceptable organic nitrogen-containing base by addition of a proton, or it may be alkyl, aryl or aralkyl; M may be a pharmaceutically acceptable alkaline earth metal ion; and R and R when taken separately may be alkyl, aryl or aralkyl or taken together with the nitrogen to which they are attached, may form a heterocyclic ring.

M may, for example, be sodium, potassium, lithium, mono-, dior trialkylammonium in which the alkyl groups contain up to 12 carbon atoms; mono-, dior trialkanolammonium in which the alkanol groups contain from 2 to 4 carbon atoms; it may be piperidinium, pyrrolidinium, morpholinium, pyrrolinium or N-alkyl derivatives thereof in which the alkyl group contains up to 3 carbon atoms; or it may be alkyl containing from 1 to 18 carbon atoms, phenyl or phenyl alkyl in which the alkyl group contains up to 4 carbon atoms; M may be calcium or magnesium; and R and R may be alkyl containing from 1 to 18 carbon atoms, hydroxyalkyl containing from 2 to 4 carbon atoms, phenyl, phenylalkyl in which the alkyl group contains up to 4 carbon atoms, or taken together with the nitrogen atom to which they are a tached R and R may represent l-piperidyl, l-pyrrolidyl, l-morpholinyl, or 1- pyrryl groups or the like.

Some of these compounds are available commercially, while others are readily prepared by methods well known to those skilled in the art. For example, they may be prepared by reaction of a secondary amine with carbon disulfide. Where two moles of the organic base are employed the corresponding amine salt is recovered as a precipitated product. Such reactions are conveniently conducted in ether, and it is generally desirable to cool the reaction mixture. If an inorganic salt is desired, an equimolar combination of the corresponding inorganic base, the sec-. ondary amine, and carbon disulfide will generally be successful. For such reactions aqueous media will be preferred. The esters and other amine salts are also readily prepared by standard methods.

The successful tumor-inhibitory activity herein reported was obtained in spite of prior teaching to the contrary, which might have been expected to discourage further study. Thus, it has been reported (Cancer Research, Sup plement I, 1953, page 107) that the benzyl ester of dibutyl dithiocarbamate failed to demonstrate activity in standard cancer screening tests.

' It has now been discovered, however, that such substances are actually surprisingly effective in inhibiting the growth of malignant tumors.

The activity of these compounds is particularly pronounced against human carcinoma cells (strain He La) grown in tissue culture according to the procedure described by Rightsel et al. (Journal of Immunology, vol. 76, pages 46774, 1956). In this test the described compounds are lethal to cancer cells at very low concentrations. For example, the sodium salt of diethyldithiocarbamic acid completely destroys all cancer cells at concentrations as low as 0.5-2.0 mcg. per ml., and the diethylammonium salt is lethal at 0.22.0 mcg. per ml. Similarly, the piperidine salt of 1-piperidinecarbodithioic acid and the pyrrolidine salt of l-pyrrolidinecarbodithioic acid are both lethal at a concentration of 3 mcg. per ml.

The compounds of the present invention exhibit significant anti-tumor activity against Crocker Sarcoma in mice. According to the procedure described by Reilly et al. (Cancer Research, vol. 13, No. 9, pages 684-7, September 1953) the substance under test is dissolved in sterile 0.85 aqueous saline. Small, uniformly cut pieces of seven-day old tumor 8-180 are implanted subcutaneously in the axillary region of Swiss white mice weighing 18 to 22 g. Each animal receives one implant, and the animals are divided into groups of six each. Intraperitoneal administration of the solution under test in doses of 0.5 cc. twice daily is begun 24 hours after implantation of the tumor and continued for a total of 13 injections. One group from each donor tumor is maintained as a control and receives injections of 0.85% saline. On the 8th day after tumor implantation the surviving animals are weighed to provide a measure of the toxic effect of the drug. The animals are then sacrificed and the tumors are excised and weighed. The Weights for each group are averaged and the averages for the treated animals are expressed as percentages of the averages for the control groups. In this test it is found that the compounds of the present invention retard tumor growth to a marked degree at tolerable dosage levels.

These compounds also exhibit significant activity against mammary adenocarcinoma CA-755 according to the procedure of Gellhorn et al. (Cancer Research, Supplement III, page 38, 1955) in which treated groups of ten animals each are employed together with untreated controls. Dosage is 0.5 cc. daily in all cases. Therapy is initiated one day after transplantation of the tumor and is continued for 12 days. At the conclusion of the experiment the animals are weighed and sacrificed, and the tumors are excised and weighed. Here again, the compounds of the present invention significantly retard growth of the tumors at the tolerated dosages. Furthermore, even where the tumor is permitted to become established by delaying the initiation of treatment for 6 days, retardation of growth is achieved.

Treatment every second day, or even every third day, is likewise beneficial. In addition to intraperitoneal administration, treatment by the subcutaneous route is also efiective.

Like many carcinostatic agents, the compounds of the present invention are somewhat toxic substances. How ever, therapeutic doses can be administered without sub- 3 stantial adverse effect. relative safety of such agents may be estimated by calculating the ratio of the maximum dose tolerated by the 'host to the minimum dose lethal to the cancer cells. Such an approximation, for example, may be made in the following manner:

A maximum dose, D tolerated by mice may be defined as that dose, expressed in micrograms of agent per gram of body weight, which at least /6 of the animals survive when treated once daily by the intraperitoneal route for five successive days. Experience with a large number of therapeutic agents has indicated that man can usually tolerate about of this level, or D /ZO, without adverse eiiects. Since the body weight is approximately one-tenth blood, such a dosage, if the agent is concentrated in the bloodstream, would provide a tolerated blood level of, 10 times D /ZO or D /Z in man. This value may be compared with the minimum concentration, LD expressed in mcg. of the agent per ml. which is found to completely destroy all tumor cells in the tissue culture test. The ratio of these two values,

for most chemotherapeutic agents now available for cancer therapy ranges from a value of about 5 to a value of about 25. In the case of the well known carcinostatic agent, Actinomycin D, this ratio has been determined to have a value of about 2.

The maximum dose of sodium diethyldithiocarbamate tolerated by mice in the test described above is found to' be about 1500 mcg./g., and the tissue culture studies previously described show that a level of 1 meg/ml. completely destroys all tumor cells. D /Z to LD for this compound has the remarkably high value of 750. Although the long term effect of the compounds of the present invention in humans will not be known for some time, their proven capability of inhibiting tumors in animals together with their relatively high therapeutic indices, calculated as discussed above, strongly indicates their value in the therapy of malignant tumors such as sarcomas, lymphomas and carcinomas in human beings.

While these compounds may beadrninistered orally, treatment by the parenteral route will usually be preferred. The substances may be employed in aqueous solution or dissolved in physiological saline, but various pharmaceutical preparations can be advantageously compounded which contain the active substance along with liquid or solid diluents. Solid preparations for extemporaneous dilution may be formulated employing various buffering agents as well as local anesthetics and other medicinal agents such as antibiotics, hypnotics, analgesics, etc., and inorganic salts to afford desirable pharmacological properties to the composition.

Doses of the order of 100 to 2000 mg./kg. daily of the compounds of the present invention are highly effective in inhibiting tumors in animals, and in human patients doses in the range of at least about 1 to 100 nag/kg. will be desirable. Therefore, the concentration of the active ingredient inthe carrier will usually be at least about 0.1%. Since these active substances are stable and widely compatible, they may be administered in solution or suspension in a variety of pharmacological- 1y acceptable vehicles, including water, propylene glycol, diethyl carbonate, glycerol, or oils such as peanut oil or sesame'oil. I

'In addition to the conventional intramuscular, subcutaneous, intraveneous, and intraperitoneal administration routes, these compounds may also be. employed in conjunction with perfusion procedures, wherein the tumor site is isolated from the main circulatory system for treatment. .Their particular usefulness vin this procedure is indicated by their especially high efiectiveness against tumor cells in-tissue culture.

Thus, the ratio As' a first approximation, the

In some cases, particularly at the :higher dosage levels, it'may be desirable to employ combinations of some of the compounds of this invention. For example, in order to avoid sodium imbalance, combinations of the sodium salt with the calcium or potassium salt or with the salt of an organic base may be advantageously utilized for cancer therapy. In addition, these active substances may be employed in combination with one or more other carcinostatic agents. For this purpose, compositions containing from 10 to of the compounds of the present invention are useful. Known carcinostatic agents which may be employed in such combinations include the nitrogen mustard type carcinostats, 6-mercaptopurine, 8-azaguanine, urethane, 6-diazo-5-oxo-l-norleucine, azaserine, triethylenemelamine, mitomycin c, triethylenephosphor amide, l,4-dimethylsulfonyloxybutane, the carcinostatic folic acid analogs, ethyl carbamate, and the like.

The following examples are provided by way of illustration, and are not intended to limit this invention, the scope of which is indicated by the, appended claims.

. EXAMPLE I I Sodium diethyldithiocarbamate'is tested for efiectiveness in inhibiting the growth of adenocarcinoma CA755 according to the procedure of Gellhorn et a1. (loc. cit.). Administration is by the intraperitoneal route in each case. Results obtained are given in Table 1;

Table' 1 Body Wt. Survival Tumor Wt. Dosage mg./ Change, g. Rate, treated treated/conkg. treated/ animals trol, percent control EXAMPLE H The experiment of Example I is repeated, with various modifications, to determine the effect of less frequent tumor therapy, the eflect of permitting the tumor to become established for 6 daysbefore treatmentis instituted, and the elfect of subcutaneous administration, with results as given inTa-ble 2. Dosage levelis 1000- mg./kg. in

Sodium diethyldithiocarbamate is evaluated for its eflectiveness in inhibiting the growth of Sarcoma following the procedure of Reilly et al. (loc. cit.) Results are as given in Table 3.

Table 3 Body-,Wt. Survival. Tumor Wt. DosagemgJ Change, g. Rate,treated.treated/con-- kg. treated/ animals trol; percent GOIIJIIOI:

EXAMPLEIV The diethylammonium salt of diethyldithiocarbamate is evaluated for effectiveness against adenocarcinoma.

CA755 according to the procedure of Example I. At a dosage level of 500 mg./ kg. average tumor Weight in the treated animals is 67% of average tumor weight in the controls. 70% of the treated animals survive, with no change in body weight, while the controls show an average weight gain of 2 grams.

EXAMPLE V The following substances are tested for their ability to retard the growth of malignant tumors according to the procedures of Examples 1 and Ill, and are found to be effective.

Piperidinium l-piperidinecarbodithioate Pyrrolidinium l-pyrrolidinecarbodithioate Morpholinium lmorpholinecarbodithioate Pyrrolinium l-pyrrolecarbodithioate N-ethylpyrrolinium N,N-diethy1dithiocarbamate Calcium N,N-diethyldithiocarbamate Magnesium N,N-dimethyldithiocarbamate Potassium N,N-distearyldithiocarbamate Lithium N,N-diphenyldithiocarbamate Methyl N,N-di(2-phenyl ethyl)dithiocarbamate Stearyl N-methyl N-ethyl dithiocarbamate Phenyl N,N-diethyldithiocarbamate Benzyl N,N-diethyldithiocarbamate 4-phenyl butyl N,N-dioctyldithiocarbamate N-dodecylammonium N,N-diethyldithiocarbamate N,N-dibutylammonium N',N'-dibutyldithiocarbamate N,N,N-trimetbylammonium N',N'-dibenzyldithiocarbamate N-ethanolammonium N'-methyl N-phenyl dithiocarbamate N,N-diethanolammonium N,N-di(Z-hydroxyethyDdithiocarbamate N,N,N-triethanolammonium N',N-diethyldithiocarbamate EXAMPLE VI A 0.1% solution of sodium diethyldithiocarbamate is aseptically prepared using isotonic saline as the vehicle. This solution is satisfactory for parenteral use in the treatment of tumors.

What is claimed is:

1. A process for retarding the growth of malignant tumors which comprises administering to a malignant tumor bearing host a daily dosage of from about 1 to 2000 mg. per kilogram of body weight of a substance represented by a formula selected from the group consisting of wherein M is selected from the group consisting of sodium, potassium, lithium, alkyl containing from 1 to 18 carbon atoms, phenyl, phenyl alkyl wherein the alkyl group contains from 1 to 4 carbon atoms; monoalkylammonium, dialkylammonium and trialkylammonium wherein the alkyl groups contain from 1 to 12 carbon atoms; alkanolammonium, dialkanolammonium, and trialkanolammonium wherein the alkanol groups contain from 2 to 4 carbon atoms; piperidinium, pyrrolidinium, morpholinium and pyrrolinium, and N-alkylderivatives thereof wherein the alkyl group contains from 1 to 3 carbon atoms;

M is selected from the group consisting of calcium and magnesium;

R and R when taken separately are selected from the group consisting of alkyl containing from 1 to 18 carbon atoms, hydroxyalkyl containing from 2 to 4 carbon atoms, phenyl, and phenyl alkyl wherein the alkyl group contains from 1 to 4 carbon atoms; and

R and R when taken together With the nitrogen atom to which they are attached are selected from the group consisting of l-piperidyl, l-pyrrolidyl, l-morpholinyl and l-pyrryl.

2. A process according to claim 1 wherein said substance is sodium diethyldithiocarbamate.

3. A process according to claim 1 wherein said substance is the diethylammonium salt of diethyldithiocarbamic acid.

4. A process according to claim 1 wherein said substance is the pyrrolidinium salt of l-pyrrolidinecarbodithioic acid.

5. A process according to claim 1 wherein said substance is the piperidinium salt of l-piperidinecarbodithioic acid.

6. A process according to claim 1 wherein said substance is the morpholinium salt of l-morpholinecarbodithioic acid.

References Cited in the file of this patent UNITED STATES PATENTS Hook et al. Mar. 26, 1957 OTHER REFERENCES Chabrier et al.: Ann. Pharm. Franc., vol. 14, pp. 720-8, 1956.

Stock et al.: Acta Unio Intern. Contra Cancrum, vol. 11, pp. 1948, 1955.

Mayer et al.: Experientia, vol. 12, pp. 322-3, 1956.

Sugiura.: Ann. N.Y. Acad. Sci., vol. 63, pp. 962-73, 1956.

Dorlands.: Illustrated Medical Dictionary, 23rd edition 1957, W. B. Saunders Co., Philadelphia, Pa., host, page 629; tumor, pages 1482-1483.

American Jurisprudence, Proof of Facts, Annotated, vol. 3, entry Cancer, pages 127-134, pub. by the Lawyers Co-operative Pub. Co., Rochester, N.Y., 1959. 

1. A PROCESS FOR RETARDING THE GROWTH OF MALIGNANT TUMORS WHICH COMPRISES ADMINISTERING TO A MALIGANT TUMOR BEARING HOST A DAILY DOSAGE OF FROM ABOUT 1 TO 2000 MG. PER KILOGRAM OF BODY WEIGHT OF A SUBSTANCE REPRESENTED BY A FORMULA SELECTED FROM THE GROUP CONSISTING OF 